Process of Producing Oil from Algae Using biological Rupturing

ABSTRACT

A process for production of biofuels from algae can include cultivating an oil-producing algae, extracting the algal oil, and converting the algal oil to form biodiesel. Extracting the algal oil from the oil-producing algae can include biologically rupturing cell wall and oil vesicles of the oil-producing algae using at least one enzyme such as a cellulose or glycoproteinase, a structured enzyme system such as a cellulosome, a virus, or combination of these materials.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/966,898, filed on Dec. 28, 2007, which claims the benefit of U.S.Provisional Patent Application No. 60/877,786, filed on Dec. 29, 2006,each of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to conversion of algae and otherbiomass to biofuels such as biodiesel or bioethanol. Accordingly, thepresent invention involves the fields of chemistry, biochemistry,genetic engineering, process engineering, algaculture, biofuels,mechanical engineering, and thermodynamics.

BACKGROUND OF THE INVENTION

Increased market prices for energy and fuels are driven by a number offactors including a depletion of easily accessible petroleum and naturalgas deposits, growth of emerging economies, political instabilities, andmounting environmental concerns. Increasing energy prices willeventually require a significant restructuring or replacement of aportion of fossil fuels by renewable energy technologies such asbiomass-based fuels.

Approximately 67% of the petroleum used in the United States iscurrently used in transportation. While the transportation sectoraccounts for less than 30% of total U.S. energy use, it is by far thelargest user of petroleum products, since electricity production andindustrial processes (the other major energy-using sectors) rely mostlyon coal, natural gas, nuclear, or hydroelectric energy. Most of theremaining energy use is residential, which is a mix of all the foregoingforms. Of the petroleum used, over 50% is now imported from outside theU.S. Petroleum imports are of increasing concern because of priceescalation and the large proportion of imports coming from potentiallyunreliable sources. In addition, concerns are growing that “greenhousegases” released from fossil fuels may contribute to climate changes. Foreconomic, environmental, and political reasons, therefore, it is highlydesirable to reduce the amount of fossil petroleum used, whichpractically means that an alternative fuel must be provided for thetransportation fleet, in addition to changes in the fleet composition.

Much research has been devoted to a long-term future in which thetransportation fleet is powered by hydrogen—the “hydrogen economy.”However, this goal has proven elusive and does not appear practical inthe foreseeable future. Instead, a more likely path to reducing orreplacing petroleum in transportation is the use of biofuels. Biofuelsare so named because they are produced from biological sources,primarily plant growth. Fossil fuels were also once produced bybiological processes, with the plant or animal products from manymillions of years ago accumulating in fossil forms of hydrocarbons.Almost all biological energy starts with the conversion of sunlight tocarbohydrates through photosynthesis. In essence, the use of petroleumreleases the energy of sunlight stored in the past, while biofuelproduction utilizes energy captured from the sun on a current basis.

In its simplest form, photosynthesis uses energy from the sun to convertcarbon dioxide and water from the environment into carbohydrates. It ispossible to release energy directly from some forms of thesecarbohydrates (e.g. burning wood or straw), but for moderntransportation such forms are not practical. Instead, a more practicalfuel can be produced by processing the plant carbohydrates into liquidforms giving higher energy densities and combustion processes moreacceptable to internal combustion engines—in other words, biofuels.

Many analyses have been done of the true economics of biofuel productioncompared to petroleum-based fuels, and most of these studies show thatin the absence of government subsidies current forms of biofuels aremore expensive on an equivalent-energy basis than petroleum fuels.However, the cost curves have been converging and are likely to crosswithin the next few years with the development of improved biofuelproduction processes and increasing prices of petroleum.

Economically, if future carbon credits are included in the analysis,then biofuels may be cheaper than petroleum fuels even today.Environmentally, the process of creating and releasing energy frombiofuels should be substantially carbon-neutral, since carbon from theatmosphere is stored in the fuel, then released once again when burned.With today's biofuel production, this ideal statement is not true, sincepetroleum fuels are used in the production of biofuels (primarilythrough agriculture). Nevertheless, as production processes for biofuelsimprove it will be possible to achieve much closer to carbon-neutralityand at lower cost than fossil fuels.

Engines for Transportation

The current transportation fleet uses mostly internal combustion enginesoperating either as compression ignition (diesel) engines burning dieselfuel or spark ignition engines burning gasoline. A much smaller amountof fuel is used in jet or turbine engines burning jet fuel (similar tokerosene). In the U.S., the ratio of gasoline to diesel fuel is abouttwo to one, with 120 billion gallons of gasoline and 60 billion gallonsof diesel fuel used annually. About 20 billion gallons of other fuelsare used, giving a total of approximately 200 billion gallons usedannually in transportation.

Diesel engines are 30 to 40% more efficient than gasoline engines. Thisis true because diesel engines operate at higher pressures (highercompression) and higher combustion temperatures than gasoline engines.If all gasoline engines were replaced with diesel engines the amount offuel needed in total would be reduced from 200 billion gallons toapproximately 160 billion gallons by this step alone.

This is a practical step to reduce petroleum use which uses currentlyavailable technologies, and will therefore likely occur worldwide. Theprocess is already well advanced in Europe and Japan. In Europe, morethan 50% of the new light vehicle fleet is diesel, compared with a muchlower percentage in the U.S. In the U.S., passenger car drivers havetraditionally avoided diesels for a number of reasons, and some states,notably California, have created regulations which make dieselsunattractive, in order to reduce emissions associated with dieselengines, including soot, nitrogen oxides, sulfur and “diesel smell.” Inaddition, most drivers consider diesels to be noisy, rough, heavy, lesspowerful, and more expensive than gasoline engines.

To some extent these complaints have been true, but new technology issolving many of these problems. For example, the growing use ofcommon-rail fuel systems results in cleaner combustion, more power, lessnoise, and smoother operation. Ultra-low-sulfur fuels are also beingintroduced, which will remove most sulfur emissions. Nitrogen oxideswill be substantially reduced in new engines in the next few years byimproved catalytic converters. Further, as will be discussed below,biodiesel burns cleaner and with lower emissions than even the mostadvanced petroleum-based diesel fuels, further tilting the balancetoward diesel engines as biodiesel production increases.

With these and other developing technologies a very attractivediesel-electric vehicle with excellent driving characteristics (superiorto most vehicles today) could replace current propulsion technologies,while providing much higher operating efficiencies. The efficienciescould double the mileage of current gasoline-engine vehicles while notsacrificing power, comfort, acceleration, or drivability.

Even if these changes in the transportation fleet do not take place,diesel fuel will still be required in very large quantities for theforeseeable future. In addition, the production process for biodiesel isgenerally more efficient than the production process associated withbioethanol or other alcohols, which is another currently availablebiofuel. Based on this analysis, the most desirable fuel for the futureis diesel fuel, and emphasis should be placed on biodiesel production.

Current Biofuel Sources

By far the largest volume of biofuel used today is in the form ofbioethanol for spark-ignition engines, with a smaller amount in the formof biodiesel for compression-ignition engines. World production ofbioethanol and biodiesel is shown in Table 1.

TABLE 1 Primary World Production of BioFuels in 2004 Production by Biofuel Type Region Feedstock Volume Bioethanol Brazil Sugarcane 5 billiongallons USA Corn 4 billion gallons EU Sugarbeet 1 billion gallonsBiodiesel Germany Rapeseed 600 million gallons USA Soybeans 50 milliongallons

Both bioethanol and biodiesel are produced primarily from plants. Theplant material used for ethanol is a form of sugar in Brazil and the EU,and corn in the U.S. For biodiesel, the primary source is oil fromrapeseed in Germany or soybeans in the U.S. The reason why these sourcesare used is that they are well known and already grown, and because thesugar, starch, or oil is relatively easy to extract and process intofuel.

However, in the long term using food crops for fuel is not optimal. Foodcrops require premium land, abundant water, and large inputs of energyin the form of agricultural machinery and fertilizer. In addition,forest land may be cleared to grow these crops, thus further depletingan already diminishing environmental resource. Competition for foodinputs will only increase, and in the event of food shortages, fuel forvehicles would become expensive. Moreover, fuel yields of these cropsare low enough that unrealistic amounts of land would be needed tosignificantly or completely replace fossil fuels.

SUMMARY OF THE INVENTION

The present invention addresses the above-described problems in novelways by using algae to produce lipids (oil) which can be readilyconverted into biodiesel through rupturing the cell wall and oilvesicles of the algae.

In accordance with one aspect, a process for production of biofuels fromalgae can include cultivating an oil-producing alga, extracting thealgal oil, and converting the algal oil to form biodiesel. Extractingthe algal oil from the oil-producing algae can include biologicallyrupturing cell wall and oil vesicles of the oil-producing algae using atleast one cellulase, glycoproeteinase, cellulosome, virus, orcombination thereof.

Additionally, a system for production of biodiesel from algae caninclude algae growth reservoirs, an oil extraction bioreactor connectedto the growth reservoir, a biological agent source connected to the oilextraction bioreactor, and a conversion reactor operatively connected tothe oil extraction bioreactor.

Additional features and advantages of the invention will be apparentfrom the following detailed description which illustrates, by way ofexample, features of the invention.

BRIEF DESCRIPTION OF THE DRAWING

Aspects of the invention can be better understood with reference to thefollowing drawing.

FIG. 1 is a process flow diagram of a system for production of biofuelsfrom algae in accordance with an embodiment of the present invention.

DETAILED DESCRIPTION

Before particular embodiments of the present invention are disclosed anddescribed, it is to be understood that this invention is not limited tothe particular process and materials disclosed herein as such may varyto some degree. It is also to be understood that the terminology usedherein is used for the purpose of describing particular embodiments onlyand is not intended to be limiting, as the scope of the presentinvention will be defined only by the appended claims and equivalentsthereof.

In describing and claiming the present invention, the followingterminology will be used.

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a step” includes reference to one or more of such steps.

As used herein, “reaction” is intended to cover single step andmulti-step reactions which can be direct reactions of reactants toproducts or may include one or more intermediate species which can beeither stable or transient.

As used herein, “biofuels” refers to any fuel, fuel additive, aromatic,and/or aliphatic compound derived from a biomass starting material suchas algae, corn, switchgrass, or the like.

As used herein, “biologically rupturing” refers to any process whichuses a biological agent to damage algae cell walls and/or oil vesiclewalls which results in a degradation, destruction or loss of integrityto such walls sufficient to allow oil materials to flow therefrom.Biological agents can be any biochemical material which has the desiredeffect such as, but not limited to, enzymes, viruses, or the like.

As used herein, “transesterify,” “transesterifying,” and“transesterification” refer to a process of exchanging an alkoxy groupof an ester by another alcohol and more specifically, of convertingalgal oil, e.g. triglycerides, to biodiesel, e.g. fatty acid alkylesters, and glycerol. Transesterification can be accomplished by usingtraditional chemical processes such as acid or base catalyzed reactions,or by using enzyme-catalyzed reactions.

As used herein, “substantial” when used in reference to a quantity oramount of a material, or a specific characteristic thereof, refers to anamount that is sufficient to provide an effect that the material orcharacteristic was intended to provide. The exact degree of deviationallowable may in some cases depend on the specific context. Similarly,“substantially free of” or the like refers to the lack of an identifiedelement or agent in a composition. Particularly, elements that areidentified as being “substantially free of” are either completely absentfrom the composition, or are included only in amounts which are smallenough so as to have no deleterious effect on the composition.

As used herein, a plurality of items, structural elements, compositionalelements, and/or materials may be presented in a common list forconvenience. However, these lists should be construed as though eachmember of the list is individually identified as a separate and uniquemember. Thus, no individual member of such list should be construed as ade facto equivalent of any other member of the same list solely based ontheir presentation in a common group without indications to thecontrary.

Concentrations, amounts, and other numerical data may be presentedherein in a range format. It is to be understood that such range formatis used merely for convenience and brevity and should be interpretedflexibly to include not only the numerical values explicitly recited asthe limits of the range, but also to include all the individualnumerical values or sub-ranges encompassed within that range as if eachnumerical value and sub-range is explicitly recited. For example, aweight range of about 1% to about 20% should be interpreted to includenot only the explicitly recited concentration limits of 1% to about 20%,but also to include individual concentrations such as 2%, 3%, 4%, andsub-ranges such as 5% to 15%, 10% to 20%, etc.

Generally, in accordance with the present invention, a process forproduction of biofuels from algae can include cultivating anoil-producing algae by promoting both autotrophic and heterotrophicgrowth. Heterotrophic growth can include introducing an algal feed tothe oil-producing algae to increase the formation of algal oil. Thealgal oil can be extracted from the oil-producing algae using biologicalagents and/or other methods such as mechanical pressing. The resultingalgal oil can be subjected to a transesterification process to formbiodiesel.

Biomass as a Biofuel Source

Rather than using high-value food crops to produce fuel, an alternativeis to use fast-growing plants that can grow on less valuable land thanfood crops, and require less input such as water, fertilizer, andpesticides compared to food crops. Since these plants do not produce thehigh-value starch and sugar of food crops, they may not be as easy touse or convert into fuels. Instead, processes can be developed whichconvert the lignocellulose or other mass of the plants into a precursor(such as starch or sugar) which can be used for fuel production.

The conversion of cellulose or other biomass into starch or sugar ismore difficult than the conversion of starch or sugar into fuel. Theconversion can be done with heat and chemicals, e.g. essentially cookingthe material to break it down, but large amounts of energy are required,which is usually supplied from fossil fuels. The cost of this step makesthe final biofuel more expensive, and also reduces the net gain inenergy because of the fuels which must be burned in the cooking stage,and also because the starch or sugar degrades somewhat in the cookingprocess and yields less energy.

Recently, biotechnology has been employed to find biological pathways tobreak down biomass into sugars or other bioavailable materials, whichcan then be fermented into alcohol or used as food for furtherbiological growth. In general such biological processes use enzymes(biological catalysts), some of which have been identified and can beproduced on an industrial scale. Such enzymes are known as cellulases.In addition, more efficient catalysts have been identified, in which aseries of enzymes is arrayed in a sequence to break down the cellulosemore rapidly and efficiently. These structured arrays are known ascellulosomes, and can be produced by certain bacteria or fungi.

With the problem of converting cellulosic mass into bioavailable feedbecoming better understood, the challenge is to produce sufficientbiomass to produce the quantity of fuels needed. A common starting pointis to begin with the food crops already used for biofuels and use theplant more efficiently. Specifically, parts of the plant other than justthe sugar or starch can be used. In the case of corn, the remainder ofthe plant other than the ears of corn is known as corn stover. In thecase of sugarcane, it is known as bagasse. By recovering and usingstover and bagasse, additional fuel can be produced from the same land.Straw from other food crops such as wheat or rice can be used in thesame way. However, even this combination of materials is not close toenough to replace the volume of fossil fuels used in transportation.

Additional plants which might be used directly or indirectly to producefuels include switchgrass, miscanthus, and certain fast-growing treessuch as hybrid willows and poplars. Switchgrass is native to NorthAmerica and can grow to ten feet tall, is perennial, grows in almostevery state, even in marginal soils, and requires little water orfertilizer. It is likely that yields of switchgrass can be improved asfurther knowledge of the species is gained. However, using currentyields of switchgrass, approximately 300 million acres would be neededto displace the current use of petroleum fuels for transportation. As areference, the total land area of the lower 48 states of the U.S. isslightly less than 3 billion acres, and approximately 1 billion acresare used for crop production or rangeland for grazing, split nearly50/50. Therefore, growing enough switchgrass would require approximatelya 60% increase in crop area. While possible, this seems impractical.

Additionally, with current processes, the biomass yields ethanol as theoutput fuel. While certainly useable as a fuel, ethanol is far fromideal. Its energy content on a volume basis is about 30% less than thefossil fuels now used, and is not practical in current diesel engines.Ethanol also attracts water, which makes storage and handling criticalto avoid exposure to water. The production process for ethanol is alsonot ideal, since energy is lost in the fermentation process. It ispossible to form alcohols other than ethanol (e.g. butanol) frombiomass, which can remove some of the disadvantages of ethanol, but thedisadvantages of the fermentation process remain. In short, replacementof petroleum fuels in transportation will need other or additional fuelsbesides alcohols.

Algae as a Biofuel Source

A search for a biosource which would require less land area than grassesor other biomass leads back to one of the first photosynthetic organismsto evolve on earth—algae. Algae can produce 10 to 100 times as much massas terrestrial plants in a year. In addition to being a prolificorganism, algae is also capable of producing oils and starches that canbe converted into biofuels.

The specific algae most useful for biofuel production are known asmicroalgae, consisting of small, often unicellular, types. These algaecan grow almost anywhere. With more than 100,000 known species ofdiatoms (a type of algae), 40,000 known species of green plant-likealgae, and smaller numbers of other algae species, algae will growrapidly in nearly any environment, with almost any kind of water.Specifically, useful algae can be grown in marginal areas with limitedor poor quality water, such as in the arid and mostly empty regions ofthe American Southwest. These areas also have abundant sunshine forphotosynthesis. In short, algae can be an ideal organism for productionof biofuels—efficient growth, needing no premium land or water, notcompeting with food crops, needing much smaller amounts of land thanfood crops, and storing energy in a desirable form.

Given the almost universal presence, ancient origins, importance, andversatility of algae, relatively little is actually known aboutsystematic cultivation of this organism. One prominent source of moderninformation has been the U.S. National Renewable Energy Laboratory(NREL), which is part of the U.S. Department of Energy (DOE). NREL,located in Golden, Colo., is responsible for research and publication ofinformation about renewable energy technologies and resources, includingbiofuel, solar and photovoltaic technology, fuel cells, hydrogen,geothermal, wind, and other energy sources.

Quoting from an NREL publication: “From 1978 to 1996, the U.S.Department of Energy's Office of Fuels Development funded a program todevelop renewable transportation fuels from algae. A primary focus ofthe program, known as the Aquatic Species Program (or ASP) was theproduction of biodiesel from high lipid-content algae grown in ponds,utilizing waste CO₂ from coal fired power plants. During the almost twodecades of this program, tremendous advances were made in the science ofmanipulating the metabolism of algae and the engineering of microalgaeproduction systems.”

Algae can store energy in its cell structure in the form of either oilor starch. Stored oil can be as much as 60% of the weight of the algae.Certain species which are highly prolific in oil or starch productionhave been identified, and growing conditions have been tested. Processesfor extracting and converting these materials to fuels have also beendeveloped but still leave much to be desired.

As described above, the primary source today for oil to producebiodiesel is the use of foodcrops such as rapeseed and soybeans. Neitherof these is likely to be able to supply a significant percentage of thetotal fuel needed for the transportation fleet. Biomass such as grasses,residue from grain crops, woodland products or waste, and so forth canpotentially supply a much larger amount of biofuels, but are almost allcurrently targeted toward the production of bioethanol. However,production of bioethanol is not optimum as a fuel source for reasonslisted above.

In accordance with one aspect of the present invention, algae is grown,its energy stores are extracted and converted into useable fuels, andbyproducts of the fuel production are either sold directly or fed backas inputs into algae growth and fuel production stages. This integratedsystem uses a minimum of external inputs other than sunlight, water, andcarbon dioxide from the air.

Building on the NREL research, other approaches propose to locate algalgrowth facilities near power plants, which are major producers of carbondioxide from combustion of fossil fuels. The otherwise wasted andpotentially harmful carbon dioxide can be fed into algae growth reactorsto increase growth, and in the process recapture carbon dioxide whichwould otherwise contribute to greenhouse gases. Despite some benefits ofthis plan, the amount of space available near power plants is limitedand therefore the amount of biofuel that can be produced this way issimilarly limited.

The present invention provides for the growth of algaephotosynthetically. That is, the primary source of energy can be the sunand the atmosphere, not the burning of fossil fuels. In one detailedaspect, the processes of the present invention can include growing twosources of algae, which may or may not be different species. Each ofthese two sources of algae may in turn be accomplished in two stages—alight stage and a dark stage. In both stages, the algae can be grown inreservoir structures, such as ponds, troughs, or tubes, which areprotected from the external environment and have controlledtemperatures, atmospheres, and other conditions. Alternatively, ifgeneric biomass is used as a feed source, it is anticipated that thiscan be purchased from growers of switchgrass or other biomass crop asdescribed above. It is believed that the present invention is the mostefficient of processes which utilize algae to form biofuels and willmake a substantial contribution to the evolution from petroleum-basedtransportation fuels to biofuels.

A novel feature of the invention is the use of algae as an intermediarystep which allows the production of biodiesel rather than bioethanol orother alcohol from generic biomass. In this aspect of the presentinvention, biomass can be depolymerized to produce sugar. The sugar isthen fed to oil-producing algae, which convert the energy content of thesugar into oil (algal triglycerides), which can then be furtherconverted into biodiesel. Most of the energy of the sugar ends up asuseful fuel, rather than suffering the fermentation losses which occurduring the production of alcohol, e.g. in the form of heat and carbondioxide emissions.

Algal Growth Stage

In accordance with the present invention, algae growth reservoirs caninclude a carbon dioxide source and a circulating mechanism configuredto circulate an oil-producing algae within the algae growth reservoirs.It was shown earlier that approximately 300 million acres ofconventional biomass growth would be needed to totally displace today'stransportation fuels. In contrast, less than 10 million acres of algaegrowth reservoirs would be needed. One way to achieve such large surfacegrowth areas is in large ponds or in a captive marine environment. Inone embodiment, a raceway pond can be used as an algae growth reservoirin which the algae is grown in shallow circulating ponds with constantmovement around the raceway and constant extraction or skimming off ofmature algae.

It is also known that certain species of algae are much more prolific inthe production of oil or starch than others. Unfortunately, thesespecies seem generally to be susceptible to predation or displacement bynative or volunteer species which exist naturally in the environmentwhere the growth reservoir is located. Moreover, in most locations,temperatures, while generally moderate, may reach extremes of heat orcold which could damage or at least retard the growth of the algae. Forthese and other reasons, some form of protection is usually desirablefor the chosen algae species. In another aspect, low-cost greenhousescan be built over the raceway ponds. These greenhouses can have enoughintegrity to maintain a positive pressure with airlocks, filtration, andtemperature control. This integrity can prevent the entrance of wildalgae and can maintain desired conditions for the algae crop.

Some species of algae important for fuel production can grow in eitherlight or dark conditions. In light conditions, growth isphotoautotrophic (or simply autotrophic), meaning that light, e.g.sunlight, provides the energy needed for growth. In dark conditions,growth is heterotrophic, meaning that an outside form of energy or foodother than light is needed. Autotrophic growth and heterotrophic growthare different growth mechanisms, and can be used in various ways in thetwo stages of the present invention to yield optimum fuel production.

Referring now to FIG. 1, an oil-producing algae can be cultivated in acultivation sub-system 10. Both autotrophic and heterotrophic growth canbe used to produce a useful quantity of algae and for the algae toproduce useful oil. In one alternative embodiment of the presentinvention, the autotrophic growth and heterotrophic growth can besequentially performed in a two-stage process. In the first stage 2, thealgae can be grown in a greenhouse environment such as the raceway pondsas described above, although other growth environments may also besuitable. Non-limiting examples of growth environments or reservoirswhich can be used include bioreactors, open ponds having various shapesand configurations, and the like. Each of these options can havebenefits and drawbacks. For example, current bioreactors areprohibitively expensive for large scale production, although futuredevelopments may make this mechanism more attractive for use inconnection with the present invention. In this first stage, the algaegrows photosynthetically, with air circulation 4 providing the carbonsource from atmospheric carbon dioxide with optional additional carbondioxide from other intra or extra-process sources, e.g. fermentation oroff-gas sequestration. The purpose of the light stage is to grow a largemass of green algae as quickly and cheaply as possible. This first-stagealgae can then be skimmed off, moved, and/or exposed immediately to thesecond stage 6, or dark stage.

The structure of the dark or autotrophic stage 6 can be very similar tothe light stage 2, except that the structure can be opaque rather thantransparent to light as in the greenhouse, thus providing nearly darkconditions for growth. The opaque conditions can be provided by amovable covering, by a separate enclosure, or through any other suitablemechanism. In the dark stage certain conditions and nutrients can beoptimized to encourage the production of starch or oil for conversion tofuels. For example, the nitrogen content of the water may be reduced,and food in the form of carbon dioxide and/or sugar can be fed into thewater in a raceway pond. Certain nutrients, especially a balanced mix ofmagnesium and potassium, can be added to the algal food supply.Optionally, genetic manipulations of key metabolic factors in the algaeto increase oil or starch production may be undertaken, or biologicalmaterials may be added to trigger or accelerate oil production.Similarly, the heterotrophic stage can be entirely or at least partiallytriggered by using a stress induction mechanism in order to shift growthfrom autotrophic to heterotrophic growth. Non-limiting examples ofsuitable stress induction mechanism can include light deprivation,nutrient deprivation (e.g. nitrogen and/or phosphorous), injection of areactive oxygen source (e.g. ozone or peroxide), and/or chemicaladditives.

These steps can be taken to provide the optimal conditions to induce thealgae to stop producing green mass and, instead, biologically convert tofilling the cell bodies with as much oil or starch as possible. Thiseffect has been conclusively demonstrated, and the underlying theory isthat the dark condition and altered nutrient availability create stressin the algae, and in preparation for possible long-term harsh conditionsthe algae store up energy in the compact form of a lipid or starch byextracting carbon and energy from the available nutrients in lieu ofgreen growth through photosynthesis.

This two-step sequence optimizes total production of the desiredsubstances by first creating as many cell bodies as possible to increasethe overall algae population, then altering the conditions to reducecreation of new cell bodies and instead fill them with oil or starch.Neither step alone would produce the desired effect useful for thepresent invention.

Generally, the oil growth reservoirs can include an algae growth controlmeans for achieving both autotrophic growth and heterotrophic growth.The algae growth control means can include a stress induction mechanismfor controlling available light and nutrient levels to the algae growthreservoir. An alternative embodiment can further include the addition ofmaterials, which will initiate and induce the production of oils orstarches directly. For example, a single algae growth reservoir can besubjected to a controllable light environment by retractable coverings,reversibly opaque coverings, or the like. Alternatively, the algaegrowth control means can include providing separate autotrophic growthreservoirs and heterotrophic growth reservoirs within the algae growthreservoirs which are operatively connected to one another and configuredfor autotrophic growth and heterotrophic growth, respectively.

Although a staged process can be used, a combined growth process can bepreferred under some circumstances. For example, the autotrophic growthand heterotrophic growth can be performed substantially simultaneouslyor overlapping. In one detailed aspect of the present invention, theautotrophic growth and heterotrophic growth are performed substantiallysimultaneously by providing a lipid trigger to initiate and/oraccelerate oil production during heterotrophic growth.

Strains of Algae

Lipid or oil-producing algae can include a wide variety of algae,although not all algae produce sufficient oil, as mentioned above. Themost common oil-producing algae can generally include, or consistessentially of, the diatoms (bacillariophytes), green algae(chlorophytes), blue-green algae (cyanophytes), and golden-brown algae(chrysophytes). In addition a fifth group known as haptophytes may beused. Specific non-limiting examples of bacillariophytes capable of oilproduction include the genera Amphipleura, Amphora, Chaetoceros,Cyclotella, Cymbella, Fragilaria, Hantzschia, Navicula, Nitzschia,Phaeodactylum, and Thalassiosira. Specific non-limiting examples ofchlorophytes capable of oil production include Ankistrodesmus,Botryococcus, Chlorella, Chlorococcum, Dunaliella, Monoraphidium,Oocystis, Scenedesmus, and Tetraselmis. In one aspect, the chlorophytescan be Chlorella or Dunaliella. Specific non-limiting examples ofcyanophytes capable of oil production include Oscillatoria andSynechococcus. A specific example of chrysophytes capable of oilproduction includes Boekelovia. Specific non-limiting examples ofhaptophytes include Isochrysis and Pleurochrysis.

In one preferred aspect, the oil-producing algae can have oil contentgreater than about 20%, and preferably greater than about 30% by weightof the algae. Currently known strains contain a practical maximum oilcontent of about 40% by weight, although levels as high as 60% have beenshown, and strains developed or discovered in the future may achievepractical maximums higher than 40%. Such strains would certainly beuseful in connection with the present invention. In some embodiments,the oil production stage algae can comprises greater than 50% or consistessentially of the oil-producing algae. Optimal heterotrophic growthtimes can vary depending on the strain of algae and other operatingparameters. This can involve a balance of oil production, feed costs,and diminishing returns of oil percentage increases. For example, algalstrains which can reach 50% oil may take only 5 days to reach 35% andanother 10 days to reach 50%. In such a case, it may be suitable toharvest the oil at five days and begin cultivating another batch ratherthan expending additional energy in growing the final 15% oil content.As a general guideline, heterotrophic growth times can range from 2 daysto 2 weeks, and are often from about 4 to 7 days. At longer times,foreign algae, bacteria and other factors can make maintainingacceptable growth conditions progressively more difficult.

Further, the oil-producing algae of the present invention can include acombination of an effective amount of two or more strains in order tomaximize benefits from each strain. As a practical matter, it can bedifficult to achieve 100% purity of a single strain of algae or acombination of desired algae strains. However, when discussed herein,the oil-producing algae is intended to cover intentionally introducedstrains of algae, while foreign strains are preferably minimized andkept below an amount which would detrimentally affect yields of desiredoil-producing algae and algal oil. Undesirable algae strains can becontrolled and/or eliminated using any number of techniques. Forexample, careful control of the growth environment can reduceintroduction of foreign strains. Alternatively, or in addition to othertechniques, a virus selectively chosen to specifically target only theforeign strains can be introduced into the growth reservoirs in anamount which is effective to reduce and/or eliminate the foreign strain.An appropriate virus can be readily identified using conventionaltechniques. For example, a sample of the foreign algae will most ofteninclude small amounts of a virus which targets the foreign algae. Thisvirus can be isolated and grown in order to produce amounts which wouldeffectively control or eliminate the foreign algae population among themore desirable oil-producing algae.

During autotrophic growth, nutrients can be supplied to theoil-producing algae. In one particular aspect, a nitrogen-fixing algaecan be introduced to the oil-producing algae to supply nitrogen as anutrient. The nitrogen-fixing algae (8 of FIG. 1) can be introduced byeither mixing and/or additional process steps to release nitrogen foruse by the oil-producing algae. In one embodiment, the nitrogen-fixingalgae can include, or consist essentially of, cyanobacteria, i.e.blue-green algae. In another embodiment, the oil-producing algae mayitself be cyanobacteria, which can then fix its own nitrogen, therebyreducing costs for the oil-producing growth.

Process to Select Strains of Algae for Optimal Growth and Oil Production

To achieve optimal oil production, three steps can be applied. The firstis the selection of species of algae with desirable characteristics ofgrowth, robustness, and high lipid production. The second is theidentification of metabolic pathways involved in triggering andincreasing lipid production, and the third is the application ofmechanisms to affect these metabolic pathways.

Balance of Oil-Production vs. Cellulose or Starch Production

While the process of the present invention can produce both biodieseland bioethanol, it is optimized for the production of biodiesel.Biodiesel production is preferable for several reasons, the first ofwhich is the higher efficiency and likely evolution of a diesel-basedtransportation fleet. The second reason is that the production of energyin the form of oil (lipids) by algae is more useful than the productionof starch. If equal volumes of oil and starch are produced, the oil willcontain significantly more energy. For example, the energy content in atypical algal lipid is 9 kcal/gram compared to 4.2 kcal/gram for typicalalgal starch. Third, in the production of sugars from starch, not allthe starch is saccharified into sugars which can be easily fermented, soa portion may be lost as unused sugars. The final reason is that theproduction of biodiesel from the algal oil is essentiallyenergy-neutral, so nearly all of the energy content of the algal oil isretained in the biodiesel. In contrast, the production of alcohol frombiomass or starch is less efficient, especially during the fermentationstage which converts the sugars derived from the biomass or starch intoalcohol. Fermentation is exothermic, with heat being generated that mustbe removed and often wasted. In addition, one half of the carbon in thesugar is released during fermentation as carbon dioxide and is thereforenot available for fuel energy. For all of these reasons biodieselproduction is more efficient overall than bioethanol production andtherefore the goal of highest efficiency and lowest cost is served bymaximizing biodiesel production.

Nevertheless, starch-producing or biomass producing algae are oneimportant aspect of the present invention, as described below in moredetail. For example, starch products or sugars converted from algalbiomass can be used to produce feed for the oil-producing algae and/orproduction of ethanol or ethyl acetate for use in transesterification ofalgal oil. Such algal biomass can be the same or different algae strainsthan those used as the oil-producing algae. If the starch or biomassgrowth is greater than needed, the excess can be converted to ethanoland sold profitably as a second product in addition to biodiesel. Carbondioxide released during fermentation can be fed back into the algalgrowth stage, substantially eliminating at least this form of energyloss in the fermentation process.

Recovery of Oil, Starch and Sugar from the Algae

Algae store oil inside the cell body, sometimes but not always invesicles. This oil can be recovered in several relatively simple ways,including solvents, heat, and/or pressure. However, these methodstypically recover only about 80% to 90% of the stored oil. The processesof the present invention offer more effective oil extraction methodswhich can recover close to 100% of the stored oil at low cost. Thesemethods include or consist of depolymerizing, such as biologicallybreaking the walls of the algal cell and/or oil vesicles, if present, torelease the oil from the oil-producing algae.

Oil can be extracted in extraction sub-system 20. The extractionsub-system can include an oil extraction bioreactor 14 operativelyconnected to the algae growth reservoirs 6, 2. Within the oil extractionbioreactor the cell walls and algal oil vesicles of the oil-producingalgae can be biologically ruptured to yield an algal oil and algalresidue. A biological agent source 12 can be operatively connected tothe oil extraction bioreactor. The processes of the present inventioncan generally use at least one of three types of biological agents torelease algae energy stores, i.e. enzymes such as cellulase orglycoproteinase, structured enzyme arrays or system such as acellulosome, a viral gent such as a virus or viral lysate, or acombination thereof. A cellulase is an enzyme that breaks downcellulose, especially in the wall structures, and a cellulosome is anarray or sequence of enzymes or cellulases which is more effective andfaster than a single enzyme or cellulase. In both cases, the enzymesbreak down the cell wall and/or oil vesicles and release oil or starchfrom the cell. Cellulases used for this purpose may be derived fromfungi, bacteria, or yeast. Non-limiting examples of each includecellulase produced by fungus Trichoderma reesei and many geneticvariations of this fungus, cellulase produced by bacteria genusCellulomonas, and cellulase produced by yeast genus Trichosporon. Aglycoproteinase provides the same function as a cellulase, but is moreeffective on the cell walls of microalgae, many of which have astructure more dependent on glycoproteins than cellulose.

In addition, a large number of viruses exist which invade and rupturealgae cells, and can thereby release the contents of the cell—inparticular stored oil or starch. Such viruses are an integral part ofthe algal ecosystem, and many of the viruses are specific to a singletype of algae. Specific examples of such viruses include the chlorellavirus PBCV-1 (Paramecium Bursaria Chlorella Virus) which is specific tocertain Chlorella algae, and cyanophages such as SM-1, P-60, and AS-1specific to the blue-green algae Synechococcus. The particular virusselected will depend on the particular species of algae to be used inthe growth process. One aspect of the present invention is the use ofsuch a virus to rupture the algae so that oil or starch contained insidethe algae cell wall can be recovered. In another detailed aspect of thepresent invention, a mixture of biological agents can be used to rupturethe algal cell wall and/or oil vesicles.

Mechanical crushing, for example, an expeller or press, a hexane orbutane solvent recovery step, supercritical fluid extraction, or thelike can also be useful in extracting the oil from oil vesicles of theoil-producing algae. Alternatively, mechanical approaches can be used incombination with biological agents in order to improve reaction ratesand/or separation of materials. Regardless of the particular biologicalagent or agents chosen such can be introduced in amounts which aresufficient to serve as the primary mechanism by which algal oil isreleased from oil vesicles in the oil-producing algae, i.e. not a merelyincidental presence of any of these.

Once the oil has been released from the algae it can be recovered orseparated 16 from a slurry of algae debris material, e.g. cellularresidue, oil, enzyme, by-products, etc. This can be done bysedimentation or centrifugation, with centrifugation generally beingfaster. Starch production can follow similar separation processes.Recovered algal oil 18 can be collected and directed to a conversionprocess 50 as described in more detail below. The algal biomass 22 leftafter the oil is separated may be fed into the depolymerization stagedescribed below to recover any residual energy by conversion to sugars,and the remaining husks can be either burned for process heat 62 or soldas an animal food supplement or fish food.

Conversion of Starch and Cellulose to Sugar (Depolymerization orSaccharification)

An algal feed can be formed from a biomass feed source as well as analgal feed source. Biomass from either algal or terrestrial sources canbe depolymerized in a variety of ways such as, but not limited tosaccharification, hydrolysis or the like. The source material can bealmost any sufficiently voluminous cellulose, lignocellulose,polysaccharide or carbohydrate, glycoprotein, or other material makingup the cell wall of the source material. Suitable algal feed can beprepared in feed production sub-system 30. In one aspect of the presentinvention, the algal feed can be provided by cultivating algae 26,including supplying any nutrients 28, and extracting the algal feedtherefrom after depolymerization. Alternatively, or in combination, thealgal feed can be provided by cultivating a non-algal biomass 24 andextracting the algal feed therefrom. Algae can be cultivated on-site andother terrestrial biomass can transported from exterior sources orgrowers. Preferably, at least some of the biomass can be cultivatedon-site in order to reduce transportation costs. Suitable non-algalbiomass can include any starch or cellulosic material such as, but in noway limited to, corn, sugarcane, switchgrass, miscanthus, grasses,grains, grass residues, grain residues, poplar or willow trees, othertrees, tree residues, bio-refuse, mixtures of these materials, and thelike.

In one embodiment of the invention, non-algal biomass can be the onlyinput source for sugar production, and algae in the oil-productionsub-system 10 essentially becomes a bioreactor to produce biodiesel fromgeneric biomass. In effect, the algae in oil-production sub-system 10becomes a conversion vehicle which converts feed sugar formed fromgeneric biomass into algal oil which can then be converted to biodieselas described below.

A feed biomass source, whether algal biomass 26, non-algal biomass 24,or a combination of the two, can be operatively connected to adepolymerizing reactor 32. The reactor can be configured for formingsugars from the feed biomass source by providing suitable operatingconditions. In the present invention, one approach is to use biologicalenzymes to depolymerize (break down) the biomass into sugars or othersimple molecular structures which can be used as feed for theoil-producing algae.

A feed separator 34 can be operatively connected to the depolymerizingreactor and the algae growth reservoirs. The feed separator can directat least a portion of the algal feed to the algae growth reservoirs 6 asa feed for the dark or heterotrophic stage of the oil-producing algae incultivation sub-system 10. The remainder of the algal feed can be sentforward to a fermentation sub-system 40 to form ethanol and/or ethylacetate, or alternatively other alcohols.

Fermentation

The fermentation stage can be conventional in its use of yeast toferment sugar to alcohol. The fermentation process produces carbondioxide, alcohol, and algal husks. All of these products can be usedelsewhere in the process and systems of the present invention, withsubstantially no unused material or wasted heat. Alternatively, ifethanol is so produced, it can be sold as a product or used to produceethyl acetate for the transesterification process. Similarconsiderations would apply to alcohols other than ethanol.

A fermentation sub-system 40 for forming ethanol and/or ethyl acetatecan include a fermentation reactor operatively connected to a sugarseparator of the feed sub-system 30. The fermentation reactor can beconfigured for production of ethanol 36 and/or ethyl acetate 38. In onepreferred aspect, both ethanol and ethyl acetate can be formed usingseparate fermentation reactors. For example, ethanol can be formed in afirst reactor 37 and at least a portion of the ethanol can be reactedwith acetic acid from a second reactor 36 to form ethyl acetate in athird reactor 38. The ethyl acetate can generally be formed in thepresence of other compounds and components such as, but not necessarilyincluded with or limited to, water, ethanol, acetic acid, etc. Afermentation separator can be operatively connected to the fermentationreactor for separating CO₂ product 46, ethanol and/or ethyl acetate 44,and biomass residues 42.

The carbon dioxide 46 can be captured and returned to either the light 2or dark stage 6 of the oil-producing algae cultivation step as a carbonsource to increase production of oil. A suitable CO₂ recycle line orother system can be used to direct the carbon dioxide accordingly.Biomass and/or algal residues 42 can be burned 62 from the steps offermenting and extracting an algal oil to produce heat which can bedistributed to any portion of the process as required, e.g. warming ofalgae reservoirs, and other process heat. At least a portion of ethanolor ethyl acetate product 44 can be used in the conversion of the algaloil to biodiesel via transesterification sub-system 50. Any excessethanol 48 can be stored after distillation and sold as bioethanol. Ifother alcohols, e.g. butanol, are produced rather than ethanol, thefermentation sub-system 40 would be adjusted accordingly.

Conversion of Algal Oil to Biodiesel

Algal oil can be converted to biodiesel through a process of directhydrogenation or transesterification of the algal oil. Algal oil is in asimilar form as most vegetable oils, which are in the form oftriglycerides. A triglyceride consists of three fatty acid chains, oneattached to each of the three carbon atoms in a glycerol backbone. Thisform of oil can be burned directly. However, the properties of the oilin this form are not ideal for use in a diesel engine, and withoutmodification, the engine will soon run poorly or fail. In accordancewith the present invention, the triglyceride is converted intobiodiesel, which is similar to but superior to petroleum diesel fuel inmany respects.

One process for converting the triglyceride to biodiesel istransesterification, and includes reacting the triglyceride with alcoholor other acyl acceptor to produce free fatty acid esters and glycerol.The free fatty acids are in the form of fatty acid alkyl esters (FAAE).

A transesterification reactor 52 of transesterification sub-system 50can be operatively connected to the oil extraction bioreactor ofextraction sub-system 20 to convert at least a portion of the algal oilto biodiesel. Transesterification can be done in several ways, includingbiologically and/or chemically. The biological process uses an enzymeknown as a lipase 54 to catalyze the transesterification, while thechemical process uses a synthetic catalyst 54 which may be either anacid or a base. The lipase-catalyzed reaction is preferable because itinvolves no harsh chemicals and produces a high-quality product in thesimplest way. Further, the use of ethyl acetate can be preferred overethanol or other alcohol in transesterification since alcohol can beexcessively damaging to enzyme activity. As such, in one embodiment ofthe present invention, transesterifying can include introducing anenzyme for converting the algal oil to biodiesel. Non-limiting examplesof suitable lipase may include, but are not limited to, those fromRhizomucor miehei, Thermomyces lanuginose, Pseudomonas fragi, andCandida cylindracea, and those described in U.S. Pat. Nos. 4,472,503 and5,316,927, which patents are incorporated herein by reference.

With the chemical process, additional steps are needed to separate thecatalyst and clean the fatty acids. In addition, if ethanol is used asthe acyl acceptor, it must be essentially dry to prevent production ofsoap via saponification in the process, and the glycerol must bepurified. The biological process, by comparison, can accept ethanol in aless dry state and the cleaning and purification of the biodiesel andglycerol are much easier. Either or both of the biological andchemically-catalyzed approaches can be useful in connection with theprocesses of the present invention.

Transesterification often uses a simple alcohol, typically methanolderived from petroleum. When methanol is used the resultant biodiesel iscalled fatty acid methyl ester (FAME) and most biodiesel sold today,especially in Europe, is FAME. However, ethanol can also be used as thealcohol in transesterification, in which case the biodiesel is fattyacid ethyl ester (FAEE). In the U.S., the two types are usually notdistinguished, and are collectively known as fatty acid alkyl esters(FAAE), which as a generic term can apply regardless of the acylacceptor used. Direct hydrogenation can also be utilized to convert atleast a portion of the algal oil to a biodiesel. As such, in one aspect,the biodiesel product can include an alkane.

The process of the present invention focuses on the use of ethanol orethyl acetate for transesterification because both substances can bereadily produced as part of the fermentation sub-system 40 rather thanfrom external sources. This further lowers cost because the ethanol orethyl acetate can be derived from plant material rather than from fossilsources. The ethanol may be used directly as the alcohol or may beconverted first to ethyl acetate to extend the longevity of the lipaseenzyme, if used.

Glycerol produced in transesterification 60 may be used as a dryingagent to dry the ethanol 56 for transesterification, and may also besold 60 as a product. To be used as a drying agent, the glycerol can befirst purified and dried. Pure dry glycerol is a chemical drying agentbecause it attracts and holds water. The ethanol produced from thefermentation described above is preferably essentially dry (anhydrous)to be used in chemical transesterification. Ethanol can only be dried toapproximately 96% by distillation because at this point the alcoholforms an azeotrope with water and the two components cannot be separatedfurther by conventional distillation. Thus, a distillation device can beoperatively connected between the fermentation separator and thetransesterification reactor. The remaining water can be removedchemically, and the dry glycerol produced from transesterification canbe used as the drying agent.

The algal triglyceride can also be converted to biodiesel by directhydrogenation. In this process, the products are alkane chains, propane,and water. The glycerol backbone is hydrogenated to propane, so there issubstantially no glycerol produced as a byproduct. Furthermore, noalcohol or transesterification catalysts are needed. All of the biomasscan be used as feed for the oil-producing algae with none needed forfermentation to produce alcohol for transesterification. The resultingalkanes are pure hydrocarbons, with no oxygen, so the biodiesel producedin this way has a slightly higher energy content than the alkyl esters,degrades more slowly, does not attract water, and has other desirablechemical properties.

Final Products

The final products from the processes presented herein are large amountsor proportions of biodiesel 58 and possibly lesser amounts orproportions of bioethanol 48 resulting from any excess production notused for biodiesel. If direct hydrogenation is used, then no alcoholwill be produced. If transesterification is used, glycerol is producedand may be sold as a byproduct.

In addition, the process of the present invention can be highlyefficient and energy-positive, meaning the energy produced in the formof fuels is far in excess of external input energy, because very littlefossil energy is used. Algae growth requires none of the heavymachinery, expensive fuels, or chemical fertilizers and pesticidesrequired by conventional agriculture. Further, the algae can beprocessed near the growth ponds, eliminating transportation costs.

Comparison of Biodiesel to Petroleum Diesel

Following is a discussion of some of the qualities and advantages ofbiodiesel compared to petroleum diesel. Biodiesel formed in accordancewith the present invention is atmospherically carbon-neutral. In otherwords, substantially all carbon released in combustion and/orfermentation was previously removed from the atmosphere during growth.Algal oil contains little or no sulfur, so production of biodiesel usingthe present invention reduces sulfur emissions, even compared to ultralow sulfur petroleum diesel. Because of better combustion efficiency,biodiesel produces about 20% less carbon monoxide upon use by aconsumer. Biodiesel burns more efficiently which results in asmoother-running engine. The equivalent cetane number of biodiesel ishigher than petroleum diesel, reducing diesel noise and knock. Biodieselproduces less soot, reducing particulate emissions by up to 75% andnearly eliminating the black smoke often associated with diesel engineexhausts. The alkyl esters or alkanes in biodiesel contain chains withnone of the ring structures found in petroleum diesel. These petroleumring structures or aromatic hydrocarbons give diesel fuel and dieselexhaust its characteristic smell. The smell of biodiesel fuel andbiodiesel exhaust is cleaner and lacks most of the familiar dieselaroma. Biodiesel also has a higher flash point near 150° C. compared topetroleum diesel at 70° C., so it is safer to store and handle.Biodiesel breaks down about four times faster in the environment, sospills are less enduring. Biodiesel has higher lubricity so fuelinjection systems and other engine components can have a longer life.Further, biodiesel can be mixed with petroleum diesels during thetransition to 100% biodiesel fuels. Importantly, conventional dieselengines do not need to be modified to use biodiesel.

Although superior in many respects, several factors can also beconsidered when using biodiesel. FAME (methyl ester) produces slightlyless power in a diesel engine (app. 5% less) than petroleum diesel.However, since the engine runs smoother when combusting biodiesel thisdrop in power is rarely noticed. Because alkly esters break down faster,it cannot be stored as long as petroleum diesel or it may degrade,though this problem does not exist with alkanes produced byhydrogenation. Biodiesel is a solvent which increases solubility of manymaterials over petroleum diesel. As a result, older fuel tanks andsystems may release accumulated rust or sludge until the fuel system iscleaned out.

Between FAEE and FAME, FAEE can be preferred for several reasons. Theextra carbon in ethyl ester compared to methyl ester increases the heatcontent of the fuel, improving mileage and making the fuel comparable topetroleum diesel. The cetane number of ethyl ester is also higher,making the fuel burn more smoothly than methyl ester. Ethyl ester hashigher flash and combustion points, making it safer to store and use.Ethyl ester produces even less black smoke and has a lower exhausttemperature than the methyl ester variant.

It is to be understood that the above-referenced arrangements areillustrative of the application for the principles of the presentinvention. Numerous modifications and alternative arrangements can bedevised without departing from the spirit and scope of integratedprocess and systems of the present invention. While the presentinvention has been shown in the drawings and described above inconnection with the exemplary embodiments(s) of the invention, it willbe apparent to those of ordinary skill in the art that numerousmodifications can be made without departing from the principles andconcepts of the invention as set forth in the claims.

1. A system for production of biodiesel from algae, comprising: a) algaegrowth reservoirs including a carbon dioxide source and a mixingmechanism configured to circulate an oil-producing algae within thealgae growth reservoirs; b) an oil extraction bioreactor operativelyconnected to the algae growth reservoirs and configured for biologicallyrupturing algal oil vesicles of the oil-producing algae to form an algaloil and algal residue; c) a biological agent source operativelyconnected to the oil extraction bioreactor including a biological agentselected from a cellulase, a glycoproteinase, a cellulosome, a virus, ora combination thereof; and d) a conversion reactor operatively connectedto the oil extraction bioreactor to convert at least a portion of thealgal oil to a biodiesel by direct hydrogenation or bytransesterification of the algal oil.
 2. The system of claim 1, whereinthe conversion reactor is configured to convert at least a portion ofthe algal oil to a biodiesel by direct hydrogenation of the algal oil.3. The system of claim 1, wherein the conversion reactor is configuredto convert at least a portion of the algal oil to a biodiesel bytransesterification of the algal oil.
 4. The system of claim 1, whereinthe biological agent includes a virus.
 5. The system of claim 4, whereinthe biological agent consists essentially of at least one virus.
 6. Thesystem of claim 1, wherein the biological agent includes a cellulase, aglycoproteinase, or cellulosome.